Stromal interaction molecule-1 (STIM1) activates store-operated Ca2+ entry (SOCE) in response to diminished luminal Ca2+ levels. Here, we present the atomic structure of the Ca2+-sensing region of STIM1 consisting of the EF-hand and sterile alpha motif (SAM) domains (EF-SAM). The canonical EF-hand is paired with a previously unidentified EF-hand. Together, the EF-hand pair mediates mutually indispensable hydrophobic interactions between the EF-hand and SAM domains. Structurally critical mutations in the canonical EF-hand, "hidden" EF-hand, or SAM domain disrupt Ca2+ sensitivity in oligomerization via destabilization of the entire EF-SAM entity. In mammalian cells, EF-SAM destabilization mutations within full-length STIM1 induce punctae formation and activate SOCE independent of luminal Ca2+. We provide atomic resolution insight into the molecular basis for STIM1-mediated SOCE initiation and show that the folded/unfolded state of the Ca2+-sensing region of STIM is crucial to SOCE regulation.

-loaded EF-SAM (holo) contains high ␣-helicity, whereas EF-SAM in the absence of Ca 2؉ (apo) is much less compact. Accordingly, the melting temperature (T m ) of the holoform is ϳ25°C higher than apoform; heat and urea-derived thermodynamic parameters indicate a Ca 2؉ -induced stabilization of 3.2 kcal mol ؊1 . We show that holoEF-SAM exists as a monomer, whereas apoEF-SAM readily forms a dimer and/or oligomer, and that oligomer to monomer transitions and vice versa are at least in part mediated by changes in surface hydrophobicity. Additionally, we find that the Ca 2؉ binding affinity of EF-SAM is relatively low with an apparent dissociation constant (K d ) of ϳ0.2-0.6 mM and a binding stoichiometry of 1. Our results suggest that EF-SAM actively participates in and is the likely the molecular trigger initiating STIM1 punctae formation via large conformational changes. The low Ca 2؉ affinity of EF-SAM is reconciled with the confirmed role of STIM1 as an ER Ca 2؉ sensor.Calcium is a fundamental signaling messenger in every eukaryotic cell, regulating a multitude of diverse and kinetically distinct cellular phenomena including gene transcription, protein folding, protein degradation, apoptosis, necrosis, and exocytosis, to name a few (1). The endoplasmic reticulum (ER) 4 is a network of folded membranes that extends through the cytoplasm to the nuclear envelope of eukaryotes. The ER membranes surround an inner cavity, the lumen, that is critical to the function of the ER as a Ca 2ϩ signaling organelle (2). Because vital Ca 2ϩ -dependent processes are associated with the ER, it is essential that changes in luminal Ca 2ϩ levels do not adversely affect these phenomena. Eukaryotes have evolved store-operated Ca 2ϩ entry (SOCE), also termed capacitive Ca 2ϩ entry, as a major Ca 2ϩ entry pathway in electrochemically non-excitable cells (3-5). SOCE is the process whereby modest ER Ca 2ϩ store depletion leads to plasma membrane (PM) Ca 2ϩ release-activated channel (CRAC) activation, providing a sustained Ca 2ϩ elevation in the cytoplasm from extracellular sources and ultimately refilling the ER luminal Ca 2ϩ stores (6). Until recently, the molecular link between ER Ca 2ϩ efflux and extracellular influx was not known. However, interfering and small inhibiting RNA studies have independently implicated stromal interaction molecule-1 (STIM1) as the likely Ca 2ϩ sensor in the ER (7,8). This single-pass, type I transmembrane protein of 685 amino acids has been found localized on both the plasma and ER membranes (3-5). The N-terminal regions of STIM1 include a signal peptide, putative EF-hand motif, and predicted sterile ␣-motif (SAM) domain. The cytosolic C-terminal region consists of two coiled-coil domains, a Pro/Ser-rich region, and a Lys-rich region (supplemental Fig. 1A) (9 -11). The putative EF-hand of STIM1 strongly aligns with the helix-loop-helix consensus sequence of this Ca 2ϩ binding motif (supplemental Fig. 1B). The EF-hand in STIM1 is somewhat unorthodox because it is seemingly unpaired, whereas Ca 2ϩ sensor pr...

The association of p120 catenin (p120) with the juxtamembrane domain (JMD) of the cadherin cytoplasmic tail is critical for the surface stability of cadherin-catenin cell-cell adhesion complexes. Here, we present the crystal structure of p120 isoform 4A in complex with the JMD core region (JMD(core)) of E-cadherin. The p120 armadillo repeat domain contains modular binding pockets that are complementary to electrostatic and hydrophobic properties of the JMD(core). Single-residue mutations within the JMD(core)-binding site of p120 abolished its interaction with E- and N-cadherins in vitro and in cultured cells. These mutations of p120 enabled us to clearly differentiate between N-cadherin-dependent and -independent steps of neuronal dendritic spine morphogenesis crucial for synapse development. NMR studies revealed that p120 regulates the stability of cadherin-mediated cell-cell adhesion by associating with the majority of the JMD, including residues implicated in clathrin-mediated endocytosis and Hakai-dependent ubiquitination of E-cadherin, through its discrete "dynamic" and "static" binding sites.

Stromal interaction molecules (STIM)s function as endoplasmic reticulum calcium (Ca 2þ ) sensors that differentially regulate plasma membrane Ca 2þ release activated Ca 2þ channels in various cells. To probe the structural basis for the functional differences between STIM1 and STIM2 we engineered a series of EF-hand and sterile α motif (SAM) domain (EF-SAM) chimeras, demonstrating that the STIM1 Ca 2þ -binding EF-hand and the STIM2 SAM domain are major contributors to the autoinhibition of oligomerization in each respective isoform. Our nuclear magnetic resonance (NMR) derived STIM2 EF-SAM structure provides a rationale for an augmented stability, which involves a 54°pivot in the EF-hand:SAM domain orientation permissible by an expanded nonpolar cleft, ionic interactions, and an enhanced hydrophobic SAM core, unique to STIM2. Live cells expressing "super-unstable" or "super-stable" STIM1/ STIM2 EF-SAM chimeras in the full-length context show a remarkable correlation with the in vitro data. Together, our data suggest that divergent Ca 2þ -and SAM-dependent stabilization of the EF-SAM fold contributes to the disparate regulation of store-operated Ca 2þ entry by STIM1 and STIM2.NMR structure | protein stability | STIM2 | store-operated calcium entry

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Forkhead box class O 3a (FOXO3a) is a transcription factor and tumor suppressor linked to longevity that determines cell fate through activating transcription of cell differentiation, survival, and apoptotic genes. Recruitment of the coactivator CBP/p300 is a crucial step in transcription, and we revealed that in addition to conserved region 3 (CR3) of FOXO3a, the C-terminal segment of CR2 (CR2C) binds CBP/p300 and contributes to transcriptional activity. CR2C and CR3 of FOXO3a interact with the KIX domain of CBP/p300 at both "MLL" and "c-Myb" binding sites simultaneously. A FOXO3a CR2C-CR3 peptide in complex with KIX exists in equilibrium between two equally populated conformational states, one of which has CR2C bound to the MLL site and CR3 bound to the c-Myb site, whereas in the other, CR2C and CR3 bind the c-Myb and MLL sites, respectively. This promiscuous interaction between FOXO3a and CBP/p300 is further supported by additional binding sites on CBP/ p300, namely, the TAZ1 and TAZ2 domains. In functional studies, our structure-guided mutagenesis showed that both CR2C and CR3 are involved in the activation of certain endogenous FOXO3a target genes. Further, phosphorylation of S626, a known AMP-dependent protein kinase target in CR3, increased affinity for CBP/p300 and the phosphomimetic mutation enhanced transactivation of luciferase. These findings underscore the significance of promiscuous multivalent interactions and posttranslational modification in the recruitment of transcriptional coactivators, which may allow transcription factors to adapt to various gene-specific genomic and chromatin structures and respond to cell signals.NMR solution structure | intrinsic disorder | promiscuous binding

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